Project Summary Environmental factors, including alcohol abuse and stress, cause long-lasting changes in the regulation of gene expression in the brain via epigenetic mechanisms, such as DNA methylation. Similar to stress, alcohol stimulates glucocorticoid release that bind to specific receptors, i.e., the glucocorticoid receptor (encoded by NR3C1). As of today, little is known on the role of epigenetic DNA modifications in regulating the transcriptome in the human prefrontal cortex (PFC, BA10) and rat PFC during chronic alcohol exposure and withdrawal. The goal of research component #4 is to interrogate genome-wide changes in DNA methylation of novel gene networks, including the NR3C1 gene network in AUD patients. Additional goals are to study whether and how altered DNA methylation and/or hydroxymethylation marks underlie the pathophysiology of AUD. The genome- wide DNA methylation approach (Infinium MethylationEPICBeadChip, Illumina) will be used in prefrontal cortex samples obtained from 30 pairs of controls and AUD subjects from the New South Wales Tissue Resource Centre (University of Sydney, Australia). In preliminary studies we identified a differential pattern of total DNA methylation in AUD for 5,254 genes. However, this technique does not differentiate 5-methyl-cytosine (5mC) from 5-hydroxymethyl-cytosine (5hmC). Here, we propose to investigate the genome?wide distribution of 5mC and 5hmC using the TET bisulfite conversion method followed by the Human MethylationEPIC BeadChip assay. Hence, we will examine the enrichment of 5hmC/5mC in association with changes in novel gene expression measured by RNA-seq. Chromatin accessibility in association with previously identified epigenetic marks will be assessed by Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). Integration of different whole-genome approaches, i.e. genome-wide DNA methylation, RNA-seq and ATAC-seq will allow an in-depth investigation of the status of the epigenome in AUD. Additionally, using a reverse-translational approach, we propose to mechanistically investigate downstream effects of DNA methylation on neuronal function in PFC and on anxiety-like behaviors and escalation of alcohol self-administration in rats treated chronically with alcohol or following a 24 h alcohol-withdrawal. Because we observed an increase of DNA methylation associated with a downregulation of TET expression (the enzyme that catalyzes the conversion of 5mC to 5hmC), we propose the use of a dCas9-Tet1-mediated protein approach to correct methylation deficits at the levels of NR3C1 and other gene promoters in the PFC of rats and determine their effect on gene expression, anxiety and drinking behaviors. The proposed study will help to identify in the human and rat brain novel epigenetic mechanisms that may provide new therapeutic targets for the treatment of AUD.